Immunoglobulin preparation

ABSTRACT

The present invention relates to the use of proline for reducing the viscosity of a protein preparation.

The present invention relates to the use of proline, and in particularL-proline, to reduce the viscosity of a protein preparation, preferablyof an immunoglobulin (Ig) preparation, more preferably of an Igpreparation comprising Ig in a mass-volume percentage of at least 18%.

The present invention further relates to a process for preparing an Igpreparation comprising Ig in a mass-volume percentage of at least 18%,to an Ig preparation obtainable by said process and to the use of saidIg preparation for preparing a medicament for the subcutaneousadministration to a human.

Primary immunodeficiency (PID) disorders, such as common variableimmunodeficiency (CVID) and X-linked agammaglobulinemia, predisposepatients to recurrent infections. These patients require immunoglobulin(Ig) replacement therapy, which can be administered intravenously (IVIg)or subcutaneously (SCIg). Immunoglobulin therapy with IVIg or SCIg hasalso been shown to be useful in the treatment of other conditions, forexample in the treatment of inflammatory and autoimmune conditions, aswell as certain neurological disorders.

If immunoglobulin is administered via the more common intravenous route,a sharp rise in serum immunoglobulin level is produced which declines asIg redistributes into the extravascular space over the next 48 hours,and then falls with first-order kinetics over approximately three weeksbefore intravenous administration is repeated. Many patients reportfeeling a “wear-off”-effect during the last week of the dosing interval,in particular malaise, fatigue, arthralgias, myalgias or increasedsusceptibility to infections.

Considering the drawbacks of intravenous Ig administration, Igadministration via the subcutaneous route has become increasinglypopular in recent years. The method does not require venous access, isassociated with only few systemic side effects and has been reported toimprove patient's quality of life.

One of the major challenges in the formulation of an Ig preparation, andin particular of an Ig preparation for subcutaneous administration, liesin the fact that Ig dissolved in aqueous solution tend to aggregate andform precipitates if not sufficiently stabilised with appropriateadditives. Carbohydrates are sometimes used as stabilisers; however,increasing concentrations of carbohydrates are associated with poortolerability, in particular in the treatment of patients with impairedkidney function (e.g. diabetes patients).

With regard to the stabilisation of monomeric Ig, particularly goodresults have been achieved by using a basic or non-polar amino acid as astabiliser. As for example disclosed in WO 2005/049078, the addition ofbasic or non-polar amino acids and the adjustment of the pH of the finalpreparation have been found to markedly decrease the formation ofaggregates and thus increase the stability of those preparations,particularly at ambient temperature.

When using the subcutaneous route for Ig-treatment of certainindications, relatively large volumes of Ig preparations need to beadministered. With the currently available Ig formulations of up to 16%(160 g/l), the inability of tissues to accept large volumes of infusedIg preparation rapidly presents a limitation to subcutaneousadministration. Thus, patients receiving Ig via the subcutaneous routeneed a relatively frequent administration of relatively small volumes atmultiple sites. Some patients and physicians regard the multiple sitesand frequent subcutaneous infusions as burdensome enough to decline orrecommend against SCIg therapy.

In view of this, Ig preparations having a higher Ig concentration wouldthus be desirable. However, an increase in the Ig concentration goesalong with a non-linear increase in viscosity which rapidly presents alimitation to the subcutaneous administration with conventional means.Specifically, highly viscous Ig preparations develop a highback-pressure and therefore compromise proper infusion by the infusionpump. In particular, a prolonged duration of administration compared topreparations having a lower concentration can be expected. This mightconsequently lead to a decrease in the acceptance of the subcutaneousroute.

Also with regard to the manufacturing process, the handling of a highlyviscous Ig preparation is relatively cumbersome.

It is thus an object of the present invention to provide a simple meansfor reducing the viscosity of a protein preparation, particularly of anIg preparation and more particularly of an Ig preparation having a highIg concentration.

It is a further object of the present invention to provide a highlyconcentrated Ig preparation, which is suitable for subcutaneousadministration and which by at least maintaining the efficacy ofcurrently available Ig preparations allows for an administration ofsmaller volumes in a fast and simple manner.

The problem is solved by the subject matter according to the independentclaims. Preferred embodiments are defined in the dependent claims.

According to a first aspect, the present invention thus relates to theuse of proline for reducing the viscosity of a protein preparation,preferably of an Ig preparation.

The term “viscosity” as used in the context of the present inventionmeans dynamic viscosity. The SI physical unit of dynamic viscosity ismillipascal second (mPs·s).

The viscosity can for example be determined by a falling ballviscosimeter (“Kugelfallviskosimeter”) according to Höppier inaccordance with the European Pharmacopoeia Version 6.0 at 2.2.49 and therequirements of DIN 53015. Thereby, the rolling time of a ball or spherein a tube or capillary of defined dimensions and having a defined slopeis determined. Based on the rolling time, the viscosity of the liquid inthe tube or capillary can be determined. The values given in the presentapplication text have been determined by the above principle using amicroviscosimeter of the type AMV200 (of Anton Paar GmbH, Graz,Austria). The measurements have been made at a temperature of 20.0° C.+/−0.1° C.

It has surprisingly been found by the present inventors that by addingproline, and in particular L-proline, a relatively low viscosity of theIg preparation can be achieved even if the concentration of Ig is high.The same effect can be achieved for other protein preparations, forexample for an albumin preparation.

Proline has been reported to have a stabilising effect on proteinpreparations, its effect of reducing the viscosity of a proteinpreparation, and in particular of an Ig preparation, has however nowherebeen considered so far.

The presence of proline thus has the double beneficial effect ofstabilising Ig on the one hand, and thus allowing to obtain apreparation having a very high stability over a relatively long periodof time, and of providing a low viscosity on the other hand, thusallowing administration of the preparation in a fast and simple manner.

As mentioned, the effect of reducing the viscosity is of particularrelevance for Ig preparations having a high Ig concentration,specifically Ig preparations having a mass-volume percentage of at least15%.

In a preferred embodiment, the Ig comprised in the Ig preparation towhich the present invention relates essentially consists of IgG. Inother preferred embodiments of the invention, the Ig comprised in the Igpreparation essentially consists of IgA or essentially consists of IgM.

In the sense of the present invention, a mass-volume percentage of 15%means 150 g per liter.

According to a second aspect, the present invention also relates to aprocess for preparing an immunoglobulin preparation comprisingimmunoglobulin in a mass-volume percentage of at least 18%, wherein saidprocess comprises the step of adding proline to reduce the viscosity ofthe preparation.

Thus, an Ig preparation having a high Ig concentration and having at thesame time a relatively low viscosity can be obtained in a very simpleand straightforward manner.

According to a particularly preferred embodiment of the process of thepresent invention, proline is added at a mass-volume percentage of theimmunoglobulin of less than 15%, preferably less than 14%, morepreferably less than 13%, and most preferably less than 12%, beforeconcentrating the preparation to the mass-volume percentage of theimmunoglobulin of at least 18%.

In contrast to the conventional process for the formulation of Igpreparations, the stabiliser—in this case proline—is added before thefinal concentration step. Thus, the protein in the concentrated productis less subject to stress conditions (e.g. shear forces) as it would bethe case if the stabiliser is added after the concentrating step. Theprocess according to this embodiment thus allows for a very gentletreatment of the product.

As set forth above, proline used in the process of the present inventionis preferably L-proline.

According to a further preferred embodiment of the process of thepresent invention, the amount of proline added is such that theconcentration of proline in the immunoglobulin preparation ranges fromabout 10 to about 1000 mmol/l, more preferably from about 100 to about500 mmol/l, and most preferably is about 250 mmol/l.

According to a particularly preferred embodiment of the process, an Igpreparation comprising Ig in a mass-volume percentage ranging from 18%to less than 20% is prepared, whereby proline is added in an amount suchthat the viscosity is less than 13 mPa·s, preferably less than 11 mPa·s,more preferably less than 10 mPa·s, and most preferably less than 9mPa·s.

According to an alternative particularly preferred embodiment, an Igpreparation comprising Ig in a mass-volume percentage of at least 20% isprepared, wherein proline is added in an amount such that the viscosityis less than 19 mPa·s, preferably less than 17 mPa·s, more preferablyless than 15 mPa·s, and most preferably less than 13 mPa·s.

Having learned from the teaching of the present invention, a skilledperson readily realizes how to choose the respective amounts of prolinein order to achieve the viscosity aimed for.

According to a third aspect, the present invention relates to an Igpreparation obtainable by the above process.

In particular, the present invention thus relates to an Ig preparationcomprising Ig in a mass-volume percentage ranging from 18% to 20%,wherein said preparation comprises proline in an amount such that theviscosity is less than 13 mPa·s, preferably less than 11 mPa·s, morepreferably less than 10 mPa·s, and most preferably less than 9 mPa·s.

More particularly, the present invention relates to an Ig preparationcomprising Ig in a mass-volume percentage of 18% to 19%, wherein saidpreparation comprises proline in an amount such that the viscosity isless than 12 mPa·s, preferably less than 11 mPa·s, and to an Igpreparation comprising Ig in a mass-volume percentage of more than 19%to less than 20%, wherein said preparation comprises proline in anamount such that the viscosity is less than 15 mPa·s, preferably lessthan 13 mPa·s.

Alternatively, the present invention also relates to an Ig preparationcomprising Ig in a mass-volume percentage of at least 20%, wherein saidpreparation comprises proline in an amount such that the viscosity isless than 19 mPa·s, preferably less than 17 mPa·s, more preferably lessthan 15 mPa·s, and most preferably less than 13 mPa·s.

More particularly, the present invention relates to an Ig preparationcomprising Ig in a mass-volume percentage of more than 20% and at themost 22%, wherein said preparation comprises proline in an amount suchthat the viscosity is less than 19 mPa·s, preferably less than 17 mPa·s,more preferably less than 14 mPa·s, most preferably less than 12 mPa·s,and to an Ig preparation comprising Ig in a mass-volume percentage ofmore than 22%, wherein said preparation comprises proline in an amountsuch that the viscosity is less than 27 mPa·s, more preferably less than20 mPa·s.

Apart from the Ig preparations defined above, the following Igpreparations can be achieved by using proline according to the presentinvention:

an Ig preparation comprising Ig in a mass-volume percentage of more than16% and at the most 17%, wherein said preparation comprises proline inan amount such that the viscosity is less than 8 mPa·s, preferably lessthan 7 mPa·s, more preferably less than 6 mPa·s; and

an Ig preparation comprising Ig in a mass-volume percentage of more than17% and less than 18%, wherein said preparation comprises proline in anamount such that the viscosity is less than 10 mPa·s, preferably lessthan 9 mPa·s, more preferably less than 8 mPa·s.

For the purpose described above, highly concentrated Ig preparations, inparticular Ig preparations comprising Ig in a mass-volume percentage ofabout 20%, are particularly preferred.

Such highly concentrated Ig preparations are preferably used for thesubcutaneous administration to patients, by way of a non-limitingexample for the treatment of PID or CVID. Preferably, the Ig comprisedin the Ig preparation of the present invention essentially consists ofIgG, as mentioned above, but is in no way limited thereto. In otherpreferred embodiments of the preparation of the present invention, theIg comprised essentially consists of IgA or essentially consists of IgM.

Given the high concentration of Ig, the present invention allows smallervolumes of the preparation to be administered to the patient, whilemaintaining the efficacy compared to conventionally availablepreparations having a lower Ig concentration.

Despite of its relatively high Ig concentration, the present inventionallows the preparation to be administered in a fast and simple mannerdue to its low viscosity. In particular, conventional means currentlyused for the conventional Ig preparations of lower concentration can beused for the subcutaneous administration.

Given its low viscosity, the Ig preparation of the present inventionallows, for example, administration by direct manual push from asyringe. The possibility to use simple devices, such as a conventionalsyringe, increases the acceptance of the subcutaneous administration andultimately lowers the cost of the treatment regimen.

Apart from its very low viscosity, the Ig preparation of the presentinvention has very high storage stability of at least 24 months whenstored at room temperature. The room temperature stability providesimproved flexibility and convenience of administration for patients withe.g. PID or CVID, compared with other preparations that must be keptrefrigerated.

As set forth above, proline is preferably L-proline. L-proline isnormally present in the human body and has a very favourable toxicityprofile. The safety of L-proline was investigated in repeated-dosetoxicity studies, reproduction toxicity studies, mutagenicity studiesand safety pharmacology studies, and no adverse effects were noted.

As also set forth above, the Ig preparation preferably comprisesproline, and in particular L-proline in a concentration ranging fromabout 10 to about 1000 mmol, preferably from about 10 to about 500mmol/l, more preferably from about 100 to about 500 mmol/l, and mostpreferably is about 250 mmol/l. L-proline used in this concentrationrange is rapidly cleared after administration of the preparation withoutany accumulation.

As sufficient stabilisation is achieved by the presence of proline, andin particular L-proline, the addition of carbohydrates as stabiliserscan be avoided. According to a preferred embodiment of the presentinvention, the preparation is thus essentially free of carbohydrates,which may have a beneficial effect on tolerability.

According to a further preferred embodiment, the Ig preparation has a pHof 4.2 to 5.4, preferably 4.6 to 5.0, most preferably about 4.8, whichfurther contributes to the high stability of the preparation.

As already stated above, an aspect of the invention is the use of asingle agent for reducing the viscosity and increasing the stability ofan immunoglobulin preparation, wherein the single agent is proline,preferably L-proline. Preferably, the amount of proline added is suchthat the concentration of proline in the immunoglobulin preparationranges from 10 to 1000 mmol/l, more preferably from 10 to 500 mmol/l,even more preferably from 100 to 500 mmol/l, most preferably about 250mmol/l.

Preferably, the immunoglobulin preparation comprises immunoglobulin in amass-volume percentage of at least 18%, more preferably at least 19%,most preferably at least 20%. Preferably, the immunoglobulin of theimmunoglobulin preparation is essentially pure IgG. Alternatively, theimmunoglobulin of the immunoglobulin preparation is essentially pure IgAor essentially pure IgM.

As given above, the advantages of the present invention are particularlyapparent if the Ig preparation is used for subcutaneous administrationto a human. The present invention thus also relates to the use of the Igpreparation for the preparation of a medicament for subcutaneousadministration to a human. As for example reported by S. Misbah et al,Clinical and Experimental Immunology, 158 (Suppl. 1); pp. 51-59, thereare various advantages of the subcutaneous administration of thepreparation over the intravenous administration. In particular, venousaccess is not required and the need for premedication withcorticosteroids and anti-histamines is reduced.

Also, when using the subcutaneous route the marked peaks typically seenwith monthly IVIg infusions are damped and persistently elevated Iglevels are obtained leading to a reduction in systemic side effects.

Due to the low viscosity of the Ig preparation of the present invention,administration can be performed in a very fast and simple manner, inparticular by direct manual push from a syringe, as mentioned above.Thus, the present invention allows self-administration of the Igpreparation by many patients at home, ultimately resulting in betterconvenience, better quality of life and fewer absences from work.

In particular, the present invention allows the preparation to beadministered by the so-called “rapid push” technique described in theabove-mentioned review article of S. Misbah et al. in the context of anIg preparation of lower concentration (Vivaglobin® comprising Ig in amass-volume percentage of 16%). According to said technique, a syringeand a 23-25-gauge butterfly needle is used to push SCIg under the skinas fast as the patient is comfortable with (usually 1 to 2 cc/min).Administration by said technique thus usually takes only between 5 and20 minutes.

A specific, non-limiting example of a process for preparing an IgGpreparation of the present invention is given in the following:

EXAMPLE Process for Preparing IgG Preparation 1. Plasma Pool

Igs were isolated from pooled human plasma derived from numerous (>1000)donors.

Starting from this suspension, the following steps were taken:

a) precipitating the human plasma using ethanol to obtain a precipitateand a supernatant;

b) subjecting the re-suspended precipitate obtained under a) to octanoicacid fractionation followed by filtration and diafiltration;

c) incubating the filtrate obtained under b) at a pH of about 4,followed by filtration;

d) subjecting the filtrate obtained under c) to anion exchangechromatography to obtain an eluate comprising Ig, said Ig comprising IgGin a purity of more than 96%;

e) subjecting the eluate obtained under d) to nanofiltration to obtain afiltrate which is essentially virus free;

f) subjecting the filtrate obtained under e) to diafiltration andultrafiltration to obtain a filtrate having a mass-volume percentage ofIgG of about 12%;

g) adding proline, and in particular L-proline, to the filtrate obtainedunder f);

h) concentrating the filtrate comprising proline to obtain an IgGpreparation having a mass-volume concentration of IgG of about 20%; and

i) adding polysorbate 80 to the IgG preparation.

The constituents and their respective amount in the final preparationare given in Table 1. Also given are the values of selectedphysicochemical parameters as well as the purity of the final Igpreparation.

TABLE 1 Composition Constituent Target Value Protein 200 g/L L-Proline*250 mmol/L (28.8 g/L) Polysorbate 80* Traces Sodium <10 mmol/LPhysicochemical properties Parameter Target Value Osmolality ~380mOsmol/kg bw pH** 4.8 (measured at 1% protein concentration in NaCl0.9%) Viscosity 14.71 mPa · s^(#) Purity Protein Typical value igG  >98% IgA <50 mg/L Monomers + dimers >90.0% *L-proline and polysorbate80 used are of non-animal origin in order to minimise the risk ofcontaminating the product with transmissible spongiform encephalopathypathogens. **To attain the optimum measurement, pH is determined in awater-diluted solution (1% protein [10 g/L] as standard). ^(#)Mean valuefrom 28 lots. Viscosity measured at room temperature

Further, the viscosity of numerous IgG preparations according to thepresent invention (comprising L-proline in a concentration of 250 mmol+/−40 mmol/l) has been determined, said preparations differing in thespecific mass-volume percentage of IgG. Some measurements with lowerproline concentrations (10 to 100mmol/l) have also been included. Theviscosity has been determined by a falling sphere viscosimeter(“Kugelfallviskosimeter”) according to Höppier in accordance with theEuropean Pharmacopoeia Version 6.0 at 2.2.49 and the requirements of DIN53015. In particular, a microviscosimeter of the type AMV200 (of AntonPaar GmbH, Graz, Austria) has been used. The measurements have been madeat a temperature of 20.0° C. +/−0.1° C.

The respective results are listed in Table 2:

TABLE 2 Mass concentration Viscosity of Proline of IgG preparationconcentration (g/l) (mPa · s) (mmol/l) 97.7 3.09 250 147.5 6.69 250173.9 7.21 250 177.5 8.79 250 178.4 7.78 250 193.7 11.15 250 198.6 11.25250 199.9 11.25 250 215.8 16.65 250 219.8 16.75 250 222.1 16.85 250 1505.7 100 143 5.8 50 144 6.0 10

In comparison, the viscosity of numerous Ig preparations devoid ofproline has been determined, the results of which being listed in Table3:

TABLE 3 Mass Viscosity of concentration preparation of IgG (mPa · s)(g/l) (without proline) 108.6 2.69 146.3 3.82 154.9 7.38 185.9 13.9194.8 16.95 207.6 21.9 227.8 34.45

According to Tables 2 and 3, the presence of proline leads to areduction in viscosity at protein concentrations higher than 15%. At amass concentration of about 200 g/l (mass-volume percentage of about20%), the viscosity of the preparation according to the presentinvention is lower than 12 mPa·s and thus far lower than the viscosityof the preparation devoid of proline.

1.-20. (canceled)
 21. A process for preparing an immunoglobulinpreparation, wherein the process comprises: (a) adding proline to aninitial immunoglobulin preparation comprising less than 15% (m/v)immunoglobulin, and concentrating the initial immunoglobulin preparationto prepare a final immunoglobulin preparation comprising at least 18%(m/v) immunoglobulin, wherein proline is added such that the viscosityof the final immunoglobulin preparation is less than 13 mPa·s asmeasured by a falling ball viscosimeter at 20.0° C. syringe, and (b)adding the final immunoglobulin preparation to a syringe, wherein theimmunoglobulin preparation in the syringe is capable of subcutaneousadministration to a human patient.
 22. The process of claim 21, whereinthe final immunoglobulin preparation comprises 100 to 500 mmol/L prolineand comprises polysorbate 80, has a pH of 4.6 to 5.0, and has aviscosity of less than 17 mPa·s as measured by a falling ballviscosimeter at 20.0° C.
 23. The process of claim 21, wherein theimmunoglobulin in the initial immunoglobulin preparation consistsessentially of IgG obtained from pooled human plasma from at least 1000donors.
 24. The process of claim 21, wherein the final immunoglobulinpreparation comprises 250 mmol/L proline.
 25. The process of claim 21,wherein the syringe is configured for subcutaneous administration to ahuman patient by direct manual push.
 26. The process of claim 21,wherein the syringe is configured for subcutaneous administration to ahuman patient by rapid push.
 27. A process for preparing animmunoglobulin preparation, wherein the process comprises: (a) addingproline to an initial immunoglobulin preparation comprising less than15% (m/v) immunoglobulin, and concentrating the initial immunoglobulinpreparation to prepare a final immunoglobulin preparation comprising atleast 18% (m/v) immunoglobulin, wherein the immunoglobulin in theinitial immunoglobulin preparation consists essentially of IgG obtainedfrom pooled human plasma from at least 1000 donors, and wherein prolineis added such that the viscosity of the final immunoglobulin preparationis less than 17 mPa·s as measured by a falling ball viscosimeter at 20.0° C., and (b) adding the final immunoglobulin preparation to a syringe,wherein the immunoglobulin preparation in the syringe is capable ofsubcutaneous administration to a human patient; and wherein the finalimmunoglobulin preparation comprises 250 mmol/L proline, comprisespolysorbate 80, and has a pH of 4.6 to 5.0.
 28. The process of claim 27,wherein the syringe is configured for subcutaneous administration to ahuman patient by direct manual push.
 29. The process of claim 27,wherein the syringe is configured for subcutaneous administration to ahuman patient by rapid push.
 30. The process of claim 27, wherein thefinal immunoglobulin preparation comprises about 20% (m/v)immunoglobulin.
 31. The process of claim 27, wherein noviscosity-lowering agents or stabilizing agents are added to the initialimmunoglobulin preparation other than proline.
 32. A process forpreparing an immunoglobulin preparation, wherein the process comprises:(a) adding proline to an initial immunoglobulin preparation comprisingless than 15% (m/v) immunoglobulin, and concentrating the initialimmunoglobulin preparation to prepare a final immunoglobulin preparationcomprising at least 20% (m/v) immunoglobulin, and wherein proline isadded such that the viscosity of the final immunoglobulin preparation isless than 19 mPa·s as measured by a falling ball viscosimeter at 20.0°C., and (b) adding the final immunoglobulin preparation to a syringe,wherein the immunoglobulin preparation in the syringe is capable ofsubcutaneous administration to a human patient; and wherein the finalimmunoglobulin preparation comprises 100 to 500 mmol/L proline,comprises polysorbate 80, and has a pH of 4.6 to 5.0.
 33. The process ofclaim 32, wherein the immunoglobulin in the initial immunoglobulinpreparation consists essentially of IgG obtained from pooled humanplasma from at least 1000 donors.
 34. The process of claim 32, whereinthe final immunoglobulin preparation comprises 250 mmol/L proline. 35.The process of claim 32, wherein the final immunoglobulin preparationhas a viscosity of less than 17 mPa·s as measured by a falling ballviscosimeter at 20.0° C.
 36. The process of claim 32, wherein thesyringe is configured for subcutaneous administration to a human patientby direct manual push.
 37. The process of claim 32, wherein the syringeis configured for subcutaneous administration to a human patient byrapid push.
 38. The process of claim 32, wherein no viscosity-loweringagents or stabilizing agents are added to the initial immunoglobulinpreparation other than proline.
 39. The process of claim 32, wherein thefinal immunoglobulin preparation is essentially free of carbohydrates.40. The process of claim 32, wherein the final immunoglobulinpreparation consists essentially of the immunoglobulin, L proline,polysorbate 80, and a sodium salt.
 41. The process of claim 32, whereinthe final immunoglobulin preparation comprises at least 20% (m/v) andnot more than 22% (m/v) immunoglobulin and wherein the immunoglobulin inthe initial immunoglobulin preparation consists essentially of IgGobtained from pooled human plasma from at least 1000 donors.
 42. Theprocess of claim 41, wherein the final immunoglobulin preparationcomprises 250 mmol/L proline.
 43. The process of claim 41, wherein thefinal immunoglobulin preparation is essentially free of carbohydrates.44. The process of claim 41, wherein the final immunoglobulinpreparation consists essentially of the immunoglobulin, L proline,polysorbate 80, and a sodium salt.